Tumors are composed of terminally differentiated and stem like cells. The latter subpopulation is believed to drive tumor growth and resistance to therapy. However, tumor stem cells are difficult to identify. No unique marker for cancer stem cells has been described and the cell of origin of tumor stem cells remains unclear. Nevertheless, tissue stem cells can be identified by surface markers or by expression of transcription factors such as Oct4, nanog, or Sox2. Based on this, we are developing fluorescence protein expressing reporter plasmids for these factors known to be expressed in tissue stem cells in order to identify stem cell subpopulation in epithelial cells, stromal cells, and tumor cells. Stem cells reside in a specialized microenvironment, the stem cell niche, which regulates stem cell maintenance, proliferation, and differentiation. Signaling molecules described in the regulation of stem cell behavior include Wnt, Notch, FGF-2, and TGF-beta. TGF-beta signals through several intracellular signaling cascades such as the Smad2/3, Smad4 and MAPK pathways, and extensively interacts with other signaling cascades such as PI3K/mTOR and BMP, and as such participates in a network that regulates differentiation, survival, proliferation and apoptosis of stem cells and differentiated cells. We plan to assess the role of these molecules in stem cell maintenance and have made strides this year in achieving this end. Heterochromatin Regulation TAZ, a transcriptional regulator, has been shown to play a role in the maintenance of pluripotency of human embryonic stem cells by controlling nucleocytoplasmic shuttling of Smad2/3-Smad4 complexes in response to TGF-beta. We have shown that inactive nuclear Smad2, 3 and 4 proteins bind to heterochromatin while the majority of inactive Smads remain in the cytoplasm. Our studies indicate that Smad3 binds specifically to centeromeric, pericenteromeric and telomeric regions of human chromosomes and that TGF-beta treatment leads to dissociation of smad3 from the two former. We plan to further investigate the role of Smads on epigenetic regulation of stem cells using 184A1 human mammary epithelial cells. These cells generate mammospheres and undergo asymmetric/symmetric division suggesting that undifferentiated 184A1 are enriched in stem cells. We plan to isolate the cells generated by symmetric and by asymmetric division and will then examine the epigenetic modification patterns in both populations to identify critical modifications in the heterochromatin and/or euchromatin structure. We have also generated the conditional Smad7 null mice to investigate the molecular/physiological mechanism of Smad7 function on stem cell maintenance. Asymmetric Cell Division Using a human breast cancer cell line we observed asymmetric activation of TGF-beta in a subset of mitotic tumor cells, implying that TGF-beta may determine the fate of daughter cells during stem cell mitosis. We are employing cortical neuronal stem cells (NSC) to investigate the influence of TGF-beta on stem cell mitosis and differentiation in collaboration with Ron McKay. We have demonstrated that NSCs endogenously express active TGF-beta, and that TGF-beta decreases NSC death in a concentration dependent fashion, while the ALK5 inhibitor, SB431542, increased cell death. Using exogenous TGF-beta and small molecule inhibitors to influence signaling cascades, we are investigating the role of the TGF-beta signaling network on NSC differentiation using high throughput imaging techniques and time lapse imaging which are established and available in the McKay lab. We aim to identify the effects of TGF-beta on stem cell differentiation and the time frame in which signaling is activated. We also plan to specifically investigate the role of Smad2/Smad3 in stem cell differentiation and maintenance. To do so we have established Smad 3 knockout colonies on BALB/c and C57/b6 backgrounds, procured Smad 3 knockout cells and tissues and established a culture of neural stem cells. We plan to establish differences in neural stem cell physiology for different mouse strains in order to obtain a baseline and then to identify differences in brain development between Smad3 wildtype and knockout mice. All methods can be readily applied to other types of stem cells as soon as those are identified. Cancer Initiation and Stem Cell Maintenance Using a a p53/Smad4/E-Cadherin conditional triple knockout breast cancer model established in collaboration with Jeff Green, we are investigating the role of Smad 4 in breast cancer initiation and stem cell maintenance. In this model system, we found that the decreased expression in each of these genes led to distinct biologic and histologic mammary tumor types. Our work also revealed a significant genetic interaction between Smad4 and E-Cadherin that appears to result in suppression of an E-Cadherin mediated adenosquamous phenotype by Smad4. Similar interactive mechanisms may occur in the development and lineage specificity of human breast cancer. We also plan to identify specific target molecules by miRNA and mRNA expression analysis using tumor tissue from these single, double, and triple knockout mice. Currently we have shown in vitro that population of Sca-1+/Lin- breast cancer cells obtained from tumor tissues of those genetically mutated mice have increased mammosphere formation and increased numbers of side population cells. The tumorigenecity of these stem-like populations is under investigation using matrigel and soft agar analysis in vitro. After confirming the tumorigenic activities, we will validate them in vivo. Furthermore, identification of cancer initiating/progenitor cells by FACS analysis using six color fluorochromes with known stem cell markers and/or other unique cell surface markers obtained from mRNA expression analysis is underway. Matrix Elasticity Finally, TGF-beta influences cell biology by altering the composition of the extracellular matrix;generally, TGF-beta increases matrix rigidity via increased stromal collagen production. It has further been shown that stem cell differentiation is strongly influenced by the matrix rigidity itself. Since matrix elasticity influences stem cell differentiation on one hand, and is involved in tumorigenesis and tumor progression in breast cancer on the other, we aim to identify whether matrix elasticity influences the tumor progression by altering the size of the tumor stem cell pool. Preliminary experiments using cell lines indicate that with increasing matrix density breast epithelial cells indeed show alterations of protein expression that are typically found in tumor cells. We are investigating whether matrix density increases malignancy in epithelial cells using the already established MCF10A model, and if this is due to the expansion of a stem cell like cell pool. We will furthermore investigate if tissue stem cell like cells can give rise to tumor cell populations if grown on a matrix of inappropriate density for the tissue.